Purification and characterization of an isozyme of catalase with enhanced-peroxidatic activity from leaves of Nicotiana sylvestris.
Two isozymes of catalase (EC 1.11.1.6), one with typically low peroxidatic activity (CAT-1) and the other with enhanced-peroxidatic activity (EP-CAT or CAT-3) have been purified to electrophoretic homogeneity from tobacco (Nicotiana sylvestris) seedlings and antibodies prepared against each. The isozyme proteins showed no immunological cross-reactivity. The subunit Mr was 55,300 +/- 750 for CAT-1 and 53,300 +/- 850 for CAT-3 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the catalatic reaction, the apparent Km values for CAT-1 and CAT-3 were 0.057 and 0.054 M, respectively, and the kcat values were 4.8 x 10(7) and 3.0 x 10(6) min-1, respectively. In the peroxidatic reaction, both have similar apparent Km's for H2O2. The apparent Km values for CAT-3 for the series methyl, ethyl, propyl, butyl, and allyl alcohols were 2.48, 5.6, 38.6, 429, and 16.3 mM, respectively. For CAT-1, the values were 697, 55.8, no detectable reaction with propyl and butyl, and 163 mM, respectively. Neither isozyme utilized dianisidine or guaiacol in the peroxidatic reaction. Catalase activity (CAT-2) which eluted in an intermediate position between CAT-1 and CAT-3 from a chromatofocusing column was composed of only one subunit whose Mr coincided with CAT-1, and only the antibody to CAT-1 reacted with CAT-2 protein. Thus, CAT-2 and CAT-1 appear closely related while CAT-3 is distinctly different.
Havir
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EA
Department of Biochemistry and Genetics, Connecticut Agricultural Experiment Station, New Haven 06504.
McHale
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D000906:Antibodies; D002374:Catalase / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010545:Peroxides / Q000378:metabolism; D010947:Plants, Toxic; D013379:Substrate Specificity; D014026:Nicotiana / Q000201:enzymology*
D000906:Antibodies; D007527:Isoenzymes; D010545:Peroxides; D002374:Catalase